SUMMARY Phage G1 gp67 is a 23 kDa protein that binds to the Staphylococcus aureus (Sau) RNA polymerase (RNAP) σA subunit and blocks cell growth by inhibiting transcription. We show that gp67 has little to no effect on transcription from most promoters but is a potent inhibitor of ribosomal RNA transcription. A 2.0-Å-resolution crystal structure of the complex between gp67 and Sau σA domain 4 (σA4) explains how gp67 joins the RNAP promoter complex through σA4 without significantly affecting σA4 function. Our results indicate that gp67 forms a complex with RNAP at most, if not all, σA-dependent promoters, but selectively inhibits promoters that depend on an interaction between upstream DNA and the RNAP α-subunit C-terminal domain (αCTD). Thus, we reveal a promoter-specific transcription inhibition mechanism by which gp67 interacts with the RNAP promoter complex through one subunit (σA), and selectively affects the function of another subunit (αCTD) depending on promoter usage.

cPromoter recognition in bacteria is mediated primarily by the subunit of RNA polymerase (RNAP), which makes sequencespecific contacts with the promoter ؊10 and ؊35 elements in the context of the RNAP holoenzyme. However, the RNAP ␣ subunit can also contribute to promoter recognition by making sequence-specific contacts with upstream (UP) elements that are associated with a subset of promoters, including the rRNA promoters. In Escherichia coli, these interactions between the RNAP ␣ subunit (its C-terminal domain [CTD], in particular) and UP element DNA result in significant stimulation of rRNA transcription. Among the many cellular and bacteriophage-encoded regulators of transcription initiation that have been functionally dissected, most exert their effects via a direct interaction with either the or the ␣ subunit. An unusual example is provided by a phage-encoded inhibitor of RNA synthesis in Staphylococcus aureus. This protein, phage G1 gp67, which binds tightly to in the context of the S. aureus RNAP holoenzyme, has recently been shown to exert selective effects on transcription by inhibiting the function of the ␣ subunit CTD (␣CTD). Here we report the development of a gp67-responsive E. coli-based transcription system. We examine transcription in vitro from promoters that do or do not carry the UP element associated with a well-characterized E. coli rRNA promoter. Our findings indicate that the ␣CTD can increase promoter activity significantly even in the absence of an UP element. We also find that gp67 can exert ␣CTD-dependent or ␣CTD-independent effects on transcription depending on the particular promoter, indicating that the mechanism of gp67 action is context dependent.